The overall objective of this project is to define the cellular mechanisms by which hormones, neurotransmitters, paracrines and growth factors interact to control hydrochloric acid secretion by parietal cells in the gastric mucosa. Gastroesophageal reflux and peptic ulcer disease are widespread clinical problems that result from or are exacerbated by gastric acid secretion. A better understanding of the mechanisms by which the major acid secretory agonists, histamine, acetylcholine and gastrin, and other factors control parietal cell acid secretion will allow fro development of improved treatment and prevention of this disease. This research should also generate useful information on the mechanisms of stimulus-secretion coupling and ion transport that are applicable to other secretory cell types including, for example, those that are affected in diseases such as cystic fibrosis and cancer. There are two major specific aims in this proposal. The first will seek to define the roles of three novel intracellular signaling proteins, lasp-1, CSPP28 and coronin/se, that have recently been identified, isolated and cloned. This aim will address the hypothesis that each of these proteins plays a specific cytoskeletal- and/or vesicle-associated role in the regulation of ion transport and that the functions of these proteins are regulated by changes in their phosphorylation. Preliminary data suggests that lasp-1 is regulated by cAMP-dependent protein kinase whereas CSPP28 is regulated by a CAM kinase and coronin/se is regulated by a calcium-independent PKC isoform. Within the context of this aim, improved DNA transfection protocols will be developed for primary cell cultures. The second aim represents a continuation of studies of intracellular signaling pathways that have been associated with the regulation of HcI secretion with an emphasis on the pathway associated with cholinergic activation of calcium-independent protein kinase C isoforms. This aim will address the hypothesis that the calcium-independent PKC isoform, PKC/epsilon, is involved in the negative regulation of HcI secretion. Little is currently known about intracellular signaling events that occur between initial agonist-receptor binding at the basolateral cell membrane and ultimate activation of ion cell membrane. Results to date suggest, however, that in all cell types second messengers activate several different protein kinases and these kinases phosphorylate a variety of cellular proteins on serine, threonine and/or tyrosine residues. The characterization of these agonist- responsive phosphoproteins as well as their upstream regulators is essential if progress is to be made in our understanding of secretory control mechanisms in parietal cells and other secretory cell types. The proposed research will employ a wide range of molecular and cellular approaches including parietal cell isolation and primary culture, cDNA transfection, phosphorylation site analyses, site directed mutagenesis, and antisense technology. Multiple video-based techniques including digitized image analysis, confocal microscopy and multi-photon microscopy will be applied in conjunction with molecular and cellular approaches to define these important intracellular signaling mechanisms.